Corosolic acid (CRA) is a substance extracted from Lagerstroemia speciosa L. and has been reported to have biological activities in in vitro and experimental animal studies. In this study, 31 subjects were orally administered 10 mg CRA or a placebo, on different occasions, in a capsule 5 min before the 75-g oral glucose tolerance test (OGTT) in a double-blind and cross-over design. Nineteen subjects had diabetes, seven had impaired glucose tolerance, one had impaired fasting glucose, and four had normal glucose tolerance according to the 1998 WHO criteria. There were no signiﬁcant differences in plasma glucose levels before and 30 min after the administration. CRA treatment subjects showed lower glucose levels from 60 min until 120 min and reached statistical signiﬁcance at 90 min. In this study, we have shown for the ﬁrst time that CRA has a lowering effect on postchalleng plasma glucose levels in vivo in humans.

Corosolic acid (CRA) is a substance extracted from Lagerstroemia speciosa L. (Banaba) and has been reported to have biological activities in in vitro and experimental animal studies (Fig. 1A). In a study using KK-Ay mice (a model of type 2 diabetes), elevation of plasma glucose levels in the group fed with a diet containing the extract from the leaf of L. speciosa was signiﬁcantly suppressed compared to the control group [1]. In studies using Ehrlich ascites tumour cells and 3T3-L1 cells, glucose uptake was stimulated by the extract fromthe leaf of L. speciosa [2,3].Recently, Judy et al. described the anti-diabetic activity of the leaf extract (including 1% CRA) in humans in a dose-dependentmanner [4]. It is known that themain content of the plant leaf extract is polyphenol, which has a blood glucose lowering effect. Screening of the compounds with blood glucose lowering activities

lagerstroemin (a kind of polyphenol) as the fraction with glucose lowering activity, but it was not enough to explain the glucose lowering effect of the total leaf extract in vivo [5]. CRA is contained in the leaf of L. speciosa, but it is still unclear whether CRA per se has an effect on a glucose challenge in humans. In this study, we have clariﬁed the effect of CRA on postchallenge plasma glucose levels in vivo in humans.

2. Subjects and methods

We examined the fasting plasma glucose (FPG) levels of 80 volunteers 2 weeks prior to the main study. The subjects who had hypertension, hepatic or renal disease, engaged in heavy exercise, or took any medication were excluded. Among them, 31 subjects (16 men and 15 women) had FPG levels between 110 and 140 mg/dl and were enrolled in this study. Thirty-one subjects were orally administered 10 mg CRA or a placebo, on different occasions, in a capsule 5 min before the 75-g oral glucose tolerance test (OGTT) in a double-blind and cross-over design. The interval between the CRA and placebo treatments was 7 days. CRA was extracted from the leaf of L. speciosa and was concentrated to a level of more than 99% after repeated high performance liquid chromatography (HPLC), as reported previously (Fig. 1B) [2]. Each 75-g OGTT was performed according to the National Diabetes Data Group recommendations, which require the

subjects to fast overnight for 10-16 h [6]. Plasma glucose and serum insulin levels were measured at baseline and 30, 60, 90, 120, and 180 min after the administration. Plasma glucose level was measured by the glucose oxidase method using a Hitachi Automatic Clinical Analyzer 7170 (Hitachi, Tokyo, Japan) and the serum insulin level was measured by enzyme immunoassay (E test Tosoh 2, Tokyo, Japan) [7,8]. Statistical analysis was performed using the Stat-View 5 system (Abacus Concepts, Berkeley, CA) [9,10].For comparison between the two treatments, paired t-test was performed, and P < 0.05 was considered statistically signiﬁcant. The data from OGTT was expressed as mean S.E.M. The studywas approved by theEthicsCommittee of Soiken Inc. andwas conducted in compliancewith theHelsinkiDeclaration [11]. All subjects gave written informed consent.

Fig. 1. (A) Structure of corosolic acid. (B) Puriﬁcation of corosolic acid from Lagerstroemia speciosa leaf extract (HPLC chart). Before the puriﬁcation, there were several peaks near the peak of corosolic acid (a). Single peak of corosolic acid was obtained after the repeated puriﬁcation of HPLC (b). (C) Effect of corosolic acid on postchallenge plasma glucose levels. Open column represents placebo treatment and closed column CRA treatment. *p < 0.05.UNCORRECTED PROOF after the administration between the two treatments. CRA treatment subjects showed lower glucose levels from 60 min until 120 min and reached statistical signiﬁcance at 90 min (Fig. 1C). Area under the curve of glucose (G-AUC) during the CRA treatment was lower than the placebo treatment, but there was no signiﬁcant difference (25482 828.1 versus 24795 911.0; mean S.E.M.). Serum insulin levels were signiﬁcantly higher at 30 min in CRA treatment subjects compared with the control subjects (32.9 4.2 mU/ml versus 27.5 3.6 mU/ml), but there were no signiﬁcant differences in serum insulin levels at other time points.

4. Discussion

In the present study, the CRA treatment subjects had lower postchallenge plasma glucose levels at 90 min than the placebo treatment subjects. Sixty minutes after the challenge, plasma glucose levels began to show a difference, and reached statistical signiﬁcance at 90 min (Fig. 1C). At 180 min, plasma glucose levels in CRA treatment subjects returned to the levels of the control. The mechanism by which CRA reduces postchallenge plasma glucose levels is not known at present. The main regulators of plasma glucose levels in humans are insulin, glucagon, and somatostatin [13–15]. Gut hormones such as GIP and GLP-1 also modulate plasma glucose levels in response to glucose challenge. It is well described that intake of polyphenols shows glucose lowering effects by the inhibition of carbohy- drate absorption. In a previous report, a negative correlation was observed between glycemic index and the concentration or total intake of polyphenols in both normal and diabetic individuals [16]. Using human intestinal cells, polyphenols were shown to decrease glucose uptake [17]. We have examined the content of an extract from the leaf of L. speciosa and detected a large amount of polyphenols (water: 5.3%, protein: 1.2%, lipid: 6.3%, carbohydrate: 73.1%, dietary ﬁber: 2.1%, and polyphenols: 10.3%). Thus, it was considered that the hypoglycemic effect of the crude extract from the leaf was an additive result of polyphenols and other factors [16–18]. In this study, we have focused on the effect of CRA per se on postchallenge glucose levels. The structure of CRA is very different frompolyphenols and other hypoglycemic agents as shown in Fig. 1A. Further studies are necessary to elucidate the mechan- ism of the hypoglycemic activities of CRA. We have described the importance of postchallenge

hyperglycemia in the mechanism of developing glucose intolerance in previous studies [19,20]. In a postchallenge state, numerous factors regulate plasma glucose levels positively and negatively with the combination of these factors determining the plasma glucose levels. At 30 min after the glucose challenge, the CRA group had higher insulin levels than the control group, a ﬁnding that may explain the lower glucose levels at 90 min. To fully elucidate the mechanism by which CRA lowers plasma glucose, further analysis of discrete organs such as liver, muscle, pancreas, and adipose tissue as well as dispersed cultured cells will be necessary. We administered CRA just before the glucose administration in the present study, as formany of the hypoglycemic agents in clinical use. It is of interest whether prolonged administration of CRA controls blood glucose levels more effectively. Pharmacological dynamics and strict dose dependence between CRA and biological responses are also to be clariﬁed. In this study, we have shown for the ﬁrst time that CRA has a lowering effect on postchallenge plasma glucose levels in vivo in humans.

Acknowledgments

This study was supported in part by Grants-in-Aid for Creative Scientiﬁc Research (10NP0201), Reserch Science and Leading Project of Biosimulation from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; a grant from ‘‘Research for the Future’’ Programof the Japan Society for the Promotion of Science (JSPS-RFTF97I00201); by Health Sciences Research Grants for Comprehensive Research on Aging and Health, Research on Health Technology Assessment, and Research on Human Genome, Tissue Engineering and Food Biotechnology from theMinistry of Health, Labour, and Welfare. We thank Use Techno Corporation, Drs. K. Yamada, Y. Fukushima, M. Izuka, and N. Tanaka for their help in the study.